The goal of this proposed research is to study the evolution of meiosis using the intestinal parasite Giardia lamblia as a model organism. Evolutionary, Giardia is the most basal eukaryote available for study having last shared a common ancestor with man or the yeasts over 1.5 billion years ago; thus it is an ideal model organism for this project. Giardia is believed to be an asexual organism, but the recent discovery of meiotic gene homologs in its genome raises the possibility that Giardia either undergoes meiosis or a process that is evolutionary related to meiosis. It has been proposed that the genes and processes involved in meiotic double stranded break repair (DSBR) and homologous recombination (HR) between homologous chromosomes evolved from processes used by somatic cells to repair DNA damage. Therefore, the presence of these candidate meiotic genes (CMGs) in Giardia allows the study of the mechanisms involved in repairing DNA damage in an evolutionary context. Understanding these mechanisms is important because mutations in many genes normally involved in DNA damage repair and meiosis lead to genome instability and cancer. The specific aims of this research are: (1) To discover molecular evidence for meiotic-like processes in Giardia. (2) To discover cytological evidence for meiotic- like processes in Giardia. (3) To search for evidence of Giardia mating both in vitro and in vivo. GFP expression data reveal that six CMGs show temporal and spatial expression patterns consistent with a meiotic-like function. The biological functions of selected CMGs involved in DNA damage/repair and nuclear fusion will be investigated by creating dominant/negative mutants. Preliminary fluorescent in situ hybridization (FISH) data support a nuclear fusion event in cysts. Additional evidence of nuclear fusion and exchange of nuclear material between nuclei will be accomplished by performing FISH on excyzoites, as well as monitoring nuclear behavior during the encystation process using both immunofluorescent microscopy and TEM. In vivo evidence of a meiotic event in Giardia will be sought with the use of a mouse model system. Because DNA damage is induced in cells undergoing meiosis, modifications of these processes can be oncogenic. If DNA repair mechanisms fail, the induction of DSBs can be deleterious to the cell and could result in genome alterations. This project will greatly enhance knowledge in the field of DNA damage repair as it relates to genome stability. Overall, this project will lead to a deeper understanding of the evolution of meiotic processes involved in DNA damage repair pathways. [unreadable] [unreadable] [unreadable]